Hi all,
in an attempt to detect multiple plasmids with a single primer pair I
thought it might be tricky to design primers for the beta-lactamase
gene (product is 800 bp). The control experiment (puc-template)
gave expected results, even though there was a light 800 bp band in
the only water control. Ok it was a dirty gel and the neighbour pocket
leaked a bit.
So I did l o o o o o t s of minipreps started PCR with our Gibco
(life-technologies) Taq and everything went wrong. I was able to
detect the 800 bp band everywhere, in pure water, in all my buffers,
in different cups, in stupid templates....
Of course I checked everything, in all combinations. Finally I lend
Taq-polymerase from a neighbour lab (Pharmacia product), and -
everything was fine. Using my buffers, templates, dNTP's I got
expected results with the Pharmacia Taq, but all positive using Gibco
Taq.
I phoned Gibco and they send me an aliquot of another Lot (this time
recombinat Enzyme) to test. This lot was OK!. I ordered more, and -
everything went wrong.
Once again we searched for a homemade contamination and again the
Enzyme (not the very lot I tested before) was the bad guy. Now we are
going to buy our Taq from Pharmacia and hope that one is clean.
By the way, Gibco said they never had any problems with their Taq.
Actually I do not think I am a genius who had this fatal primer pair
in mind for the first time in human history :).
So please, is there anyone in the Lab-world who had strange results
with beta-Lactamase primers? Any comments will be wellcome!!
Greetings from the PCR twilight zone
Georg